1. Field of the Invention
The present invention relates to a method and apparatus for a rapid disruption of cells or viruses using micro magnetic beads and a laser.
2. Description of the Related Art
Generally, a molecular diagnosis of a specific pathogenic bacteria is performed in four steps: 1) cell lysis, 2) DNA isolation, 3) DNA amplification and 4) DNA detection.
An efficient extraction of DNA from a cell is required in many applications and is essential in the molecular diagnosis, in particular, an identification and quantification of a pathogenic bacteria. The molecular diagnosis is generally performed by DNA amplification after DNA extraction. The DNA amplification includes a polymerase chain reaction (PCR), a ligase chain reaction, a stranded-displacement amplification, a nucleic acid-based amplification, a repair chain reaction, a helicase chain reaction, a QB replicase amplification, an a ligation activated transcription.
A method of isolating DNA from a cell is performed using materials having a tendency of bonding to DNA. Examples of the materials for DNA separation include silica, glass fiber, anion exchange resin and magnetic beads (Rudi, K. et al., Biotechniqures 22, 506-511 (1997); and Deggerdal, A. et al., Biotechniqures 22, 554-557 (1997)). To avoid manual steps and to remove operator error, several automatic machines are developed for high-throughput DNA extractions.
Cell lysis is conventionally performed by a mechanical, chemical, thermal, electrical, ultrasonic or microwave method (Michael T. Taylor et al., Anal. Chem., 73, 492-496 (2001)).
A chemical method includes the use of lyzing agents for disrupting cells to release DNA. An additional treatment of cell extracts with a chaotropic reagent is necessary to denature proteins. The chemical lysis method is disadvantageous in that harsh chemicals are used to disrupt the cells. Since they can interfere with the subsequent PCR, it is necessary to purify the DNA prior to the PCR. The chemical method is labor-intensive and time-consuming, requires expensive consumables and has often a low DNA yield. A thermal method involves cycles of freezing/thawing, but cannot often disrupt many structures within cells.
Heating is an alternative method of disrupting cell walls or membranes. Simple heating is disadvantageous in that it results in the denaturation of proteins, which can be attached to released DNA. They can also interfere with DNA amplification. A physical method uses a bulky and expensive pressure apparatus, which is not suitable for a Lab-on-a-Chip (LOC) application.
An ultrasonic treatment is an alternative of the physical method, wherein a cell solution or suspension is placed in a chamber located in an ultrasonic bath. Ultrasonic disruption has many disadvantages in cell lysis. First, a distribution of ultrasonic energy is not uniform. The nonuniform distribution of ultrasonic energy leads to inconsistent results. Second, due to the energy divergence in the ultrasonic bath, it takes often several minutes to completely disrupt cells. Lastly, the ultrasonic method generates unpleasant sounds.
A laser has many advantages in the disruption of cells and can be readily applied to LOC (Huaina Li et al., Anal Chem, 73, 4625-4631 (2001)).
U.S. Patent Publication No. 2003/96429 A1 discloses a laser-induced cell lysis system. When only a laser is used, an efficient cell lysis does not occur. As a result of performing an experiment using E. coli placed in a very clear solution, it is confirmed that when irradiating only a laser, a low cell lysis efficiency is obtained. A concentration of DNA measured after irradiating a laser for 150 seconds is 3.77 ng/μl because laser energy is not effectively transferred to cells. A concentration of DNA measured after boiling cells at 95° C. for 5 minutes by means of a conventional heating method is 6.15 ng/μl.
U.S. Pat. No. 6,685,730 discloses optically-absorbing nanoparticles for enhanced tissue repair. This patent includes a method of joining tissue comprising: delivering nanoparticles having dimensions of from 1 to 1000 nanometers that absorb light at one or more wavelengths to the tissue to be joined; and exposing said nanoparticles to light at one or more wavelengths that are absorbed by the nanoparticles. This method causes only a loss of function of the cells by using a laser and nanoparticles and there is no description of a method of disrupting cells by vibrating a solution containing cells and particles.
Thus, the inventors intensively studied in order to overcome the above problems and discovered that cells or viruses can be rapidly disrupted when vibrating a solution containing them using micro magnetic beads and a laser.